It is widely known in the field of microparticle analysis, e.g. flow cytometry, how to analyze cells while they are entrained by a sheath flow (i.e. buffer solution), and will now be described briefly.
When the sheath flow is disturbed, waterdrops are formed and absorb cells. Shortly before respective waterdrops escape from an adjacent portion of the flow, they are sorted out by detecting desired cells and applying electric fields to respective waterdrops. As a result, waterdrops containing desired cells are deflected by the electric fields and collected by a collection container. During this process, it is very crucial to know exactly when the waterdrops containing desired cells reach an electrified region in order to electrify specific waterdrops fully while other peripheral waterdrops are electrified slightly.
FIG. 1 shows a basic type of flow cytometry disclosed in U.S. Pat. No. 6,120,666. Referring to FIG. 1, microchannels are formed on a substrate (not shown) in a cross shape with a width small enough to allow cells 120 to pass through. The cross-shaped intersection of the microchannels acts as a focusing chamber 22, which is photographed by an image pickup device 160 (e.g. CCD camera). The resulting images are processed by an image control processor 150 so as to analyze the cells.
Particularly, a sample channel 100 extends upwards from the focusing chamber 22. Cells 120 (i.e. samples) flow in via the sample channel 100. A pair of focusing channels 102 and 104 is connected to both sides of the focusing chamber 22 so that sheath flows are introduced via them, respectively.
As shown in the drawing, a plurality of cells 120, which have been fed through the sample channel 100, are surrounded by sheath flows from both sheath flow channels 102 and 104. Then, the cells 120 pass through the focusing chamber 22 in a series so that they are photographed by the image pickup device 160 and analyzed by the image control processor 150.
The stream of the cells 120 and the sheath flows is controlled by the difference in electric potential among respective channels 100, 102, 104, and 106, which is created by applying electric fields to them.
However, the above-mentioned conventional flow cytometer, which uses electric fields so as to control cells and sheath flows, has the following problems.
The equipment necessary to apply electric fields is expensive and increases the price of the cytometer.
Since the buffer solution used as the sheath flow must be directed by electric fields, the buffer solution must be made of a conductive material. This extremely narrows the range of selectable buffer solutions.
Movement of the sheath flow based on the difference in electric potential is too slow. This lengthens the analysis time. In order to avoid this, strong electric fields of kV grade must be applied to respective channels so as to increase the difference in electric potential. However, strong electric fields unfavorably separate a specific substance (e.g. protein) from the cells. Consequently, the strength of electric fields is still limited, and the problem of slow sheath flow remains unsolved.
When microparticles have missing portions, particularly deformed red blood corpuscles, or when microparticles flow while being slanted, the scanning means (i.e. laser) is likely to pass through the missing portions or vacancies resulting from the slant. In other words, the image pickup device may fail to recognize the microparticles.
In an attempt to solve the problems occurring in the conventional flowcytometer based on electric field application, inventors of the present invention have proposed an improved apparatus as disclosed in Registered Korean Patent No. 473,362 entitled “APPARATUS AND METHOD FOR MICROPARTICLE ANALYSIS”, which will now be described with reference to FIG. 2.
As shown in FIG. 2, the apparatus includes a substrate having a focusing chamber formed thereon. At least one sample channel is formed on the substrate so as to introduce samples, particularly microparticles (e.g. cells) into the focusing chamber. At least one sheath flow channel is formed on the substrate so as to introduce a sheath flow into the focusing chamber. The sheath flow surrounds the samples and directs them in a series inside the focusing chamber. Each of the sample channel, sheath flow channel, and sorting channel is provided with a pressure control means for establishing pressure gradients with respect to other channels. The resulting pressure gradients direct the samples and the sheath flow into the focusing and shorting channels from respective channels.
Each pressure control means includes a vacuum pump connected to each container, a microvalve positioned between the vacuum pump and the container, a pressure sensor for sensing the pressure within the container, and a pressure controller for controlling the operation of the microvalve in accordance with signals from the pressure sensor.
As the samples are entrained by the sheath flow and travel in a series inside the focusing chamber due to the pressure gradients created by the pressure control means, an image pickup means photographs the samples. The resulting images are transmitted to an image controller, which counts and analyzes the samples moving inside the focusing chamber. The image controller recognizes the type of the samples and transmits corresponding information to the pressure controller for the sorting channel. As a result, the samples are sorted out according to type by corresponding pressure controllers as they pass through respective sorting channels.
As such, the improved apparatus utilizes pressure gradients instead of the difference in electric potential. Consequently, the flow rate of the sheath flow can be increased substantially without limiting the range of selectable buffer solutions. This reduces the manufacturing cost. Furthermore, there is no cell separation resulting from strong electric fields, and the reliability of cell analysis is improved remarkably. It is also possible to correctly recognize deformed microparticles, which have missing portions, and prevent microparticles from flowing while being slanted.
However, although the flowcytometer based on pressure gradients has a higher throughput due to the increased flow rate of samples and sheath flows, signals detected by detectors are too weak to guarantee an accurate sample analysis, because cells move too fast at the channel point, on which laser beams from optical units are focused, to generate a sufficient amount of fluorescence or scattering. Particularly, samples having weak fluorescence cannot be detected at all. This fatally degrades the performance of the analysis apparatus.
This problem may be avoided if the pressure of sheath flows is adjusted to slow down samples and sheath flows. This, however, substantially reduces the throughput. Although the quality of sample detection may be improved by using an optical structure having a very high resolution, the higher the resolution is, the more expensive the apparatus becomes. Furthermore, improvement of the resolution of the optical structure has its own limits.
Therefore, the present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a flowcytometer capable of detecting samples accurately even when samples and sheath flows have a high flow rate so that the quality of sample detection can be improved with no decrease in throughput.
Another object of the present invention is to provide a flowcytometer adapted to increase the resolution for sample detection if the flow rate of samples and sheath flows increases, in order to guarantee accurate sample detection regardless of the flow rate and improve the throughput.
These objects of the present invention are accomplished by expanding a portion of a focusing channel, in which samples and sheath flows combine and flow, around a channel point, on which laser beams are focused, so that sample particles slow down only in the expanded channel, thereby improving the detection intensity of sample particles. Particularly, a microchip for a flowcytometer according to the present invention has a channel expanded around a channel point, on which laser beams from optical units are focused, so that focused sample particles slow down only near the expanded channel while remaining in a focused condition. This improves the detection intensity of sample particles.
As such, the entire sample analysis throughput remains constant, because the overall flow rate of sample particles does not decrease. In addition, accurate sample analysis is guaranteed, because the sample particles slow down only in the expanded channel.
According to an aspect of the present invention, there is provided a microchip used in a flowcytometer for analyzing samples, the samples being microparticles flowing through a microchannel formed on a substrate, the microchip including a sample channel, samples flowing through the sample channel; a sheath flow channel, a sheath flow flowing through the sheath flow channel; a focusing channel, focused sample particles flowing through the focusing channel, the focused sample particles being a combination of the samples and the sheath flow; and an expanded channel formed by expanding a portion of the focusing channel, the portion being around a point for sample analysis.
According to another aspect of the present invention, there is provided a flowcytometer for analyzing samples, the samples being microparticles flowing through a microchannel, the apparatus including a focusing chamber formed on a substrate; a sample channel connected to the focusing chamber; at least one sheath flow channel connected to the focusing chamber so as to introduce a sheath flow, the sheath flow surrounding samples so that the samples flow in a series, the samples having been introduced into the focusing chamber via the sample channel; a focusing channel, focused sample particles passing through the focusing channel, the focused sample particles being a combination of the samples and the sheath flow; a pressure control means for establishing a pressure gradient between respective channels so that the samples and the sheath flow are directed to respective sorting channels via the focusing channel; and a signal detection device for detecting signals generated from samples, laser beams emitted from an optical unit being focused on the samples, wherein a portion of the focusing channel is expanded, the portion being around a point, the laser beams emitted from the optical unit being focused on the point, so that, when focused sample particles pass the expanded portion of the focusing channel, the focused sample particles slow down while remaining in a focused condition and sample particle detection intensity is improved.
The inventors of the present invention have made the following discovery: when particles have been focused into a fluid, an increase in width of a channel, through which the fluid passes, reduces the flow rate of the fluid and enlarges the width thereof. In the case of particles within the fluid, their flow rate decreases in the same manner as the fluid, but they continue to flow without being disturbed (i.e. they remain focused). Based on this discovery, it has been proposed to expand only a portion of the focusing channel so that particles slow down only in the expanded section while remaining focused. The resulting microchip for a flowcytometer can both increase the sample analysis throughput and improve the sample analysis quality.